ALBERT

Description: Key Points CD37 positivity predicts significantly better survival for DLBCL, and is superior to other prognostic factors in GCB-DLBCL. CD37 loss is an important risk factor for R-CHOP resistance in both GCB- and ABC-DLBCL.

Description: Introduction: Targeted RNA sequencing using Next Generation Sequencing (NGS) has significant advantages over transcriptome sequencing. In addition to information on mutations, fusion and alternative splicing, RNA quantification using targeted RNA sequencing is sensitive, reproducible and provides better dynamic range. We used targeted RNA sequencing for RNA profiling of diffuse large B-cell lymphoma (DLBCL) and explored its utility in the sub-classification of DLBC to ABC and GCB. Method: RNA extracted from 441 FFPE lymphnode samples with DLBC lymphoma and sequenced targeting 1408 genes. These cases were previously subclassified as ABC vs GCB using expression profiling and immunohistochemistry. We first normalized RNA expression data to PAX5 expression, then we tried to narrow down important markers using univariate significance tests. Setting the cutoff for false discovery rate at 0.0001, 48 variables remained significant, including 46 RNA levels and two genes (MYD88 and EZH2) mutation status. Using 60% of samples as training set, we used multiple statistical approaches for classification. Deep learning emerged as the best approach. We used autoencoder with 5 hidden layers and developed a model for classification of ABC vs GCB. To further improve on classification, we divided patients in each subgroup based on survival using simple tree model. In this tree model, expression level of CD58 emerged as a powerful prognostic marker for the ABC group and RLTPR expression in the GCB group. Results: Using probability of scoring developed based on deep learning and logestic regression, approximately 30% of the cases had a score between 0.5 and 0.75. For the remaining 70% of patients, the ABC vs GCB classification showed sensitivity and specificity of 96% and 97% for the testing set. We also applied the same approach to 60 independent cases classified using NanoString (Lymph2Cx). Our model showed sensitivity and specificity of 96% and 97% in the NanoString independent cases. Using the tree model for further classification of the ABC and GCB classes, CD58 mRNA levels separated the ABC group into two subgroups (ABC1 and ABC2) and RLTPR mRNA separated the GCB into two subgroups (GCB1 and GCB2) with significant difference in overall survival (P=0.0010) and progression-free survival (PFS) (P=0.0027). Conclusion: Targeted RNA sequencing is very reliable and practical for the subclassification of DLBCL and can provide clinical-grade reproducible test for prognostically subclassification of DLBCL. Figure Disclosures Albitar: Genomic Testing Ccoperative: Employment, Equity Ownership. De Dios:Genomic Testing Ccoperative: Employment. Tam:Takeda: Consultancy; Paragon Genomics: Consultancy. Hsi:Abbvie: Research Funding; Eli Lilly: Research Funding; Cleveland Clinic&Abbvie Biotherapeutics Inc: Patents & Royalties: US8,603,477 B2; Jazz: Consultancy. Ferreri:Roche: Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Kite: Consultancy. Piris:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding. Kantarjian:Ariad: Research Funding; Agios: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Novartis: Research Funding; BMS: Research Funding; Takeda: Honoraria; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Jazz Pharma: Research Funding; Cyclacel: Research Funding; Immunogen: Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Astex: Research Funding.

Description: Introduction. The majority of SMZLs display an indolent course, however the disease is still incurable and a significant proportion of patients (~25-30%) experience poor outcomes surviving 5 years, and for whom tumor material collected before initiation of medical therapy was available. Mutation analysis was performed by CAPP-seq targeted deep next generation sequencing of tumor genomic DNA. A stringent bioinformatic pipeline was applied to suppress the background noise allowing to call variants with a sensitivity of 5x10-2 in FFPE derived DNA. Copy number variations (CNVs) were identified by using the sequencing reads-based GATK4-CNV algorithm. IGHV rearrangements were obtained by using LymphoTrack® IGH FR1 Assay Panel kit. Molecular clusters were identified by an iterative algorithm that maximizes genetic distinctiveness of subgroups by reassigning patients between clusters that are created a priori based on the co-occurrence of genetic lesions. Relative survival, defined as the ratio between actuarial survival observed in the SMZL cohort and expected survival of the general population matched to patients by geographical origin, sex, age and calendar year of diagnosis, was calculated using the Ederer II method. Results. The analysis included 303 patients with a SMZL diagnosis confirmed on spleen histology. The sample size allowed to identify 30% differences in survival for molecular subgroups comprising at least 5% of cases with a statistical power between 80-100%. Median follow-up was 9.2 years. At 10 years, 85% of patients were alive, consistent with the known indolent behavior of this lymphoma. Genes recurrently affected by non-synonymous somatic mutations in 〉10% of SMZL included KLF2 (24%), NOTCH2 (19%), KMT2D (15%), TNFAIP3 (13%), EP300 and TP53 (10%). Deletion 7q was documented in 25% of cases and IGHV1-2*04 usage in 32%. By cluster analysis, three major molecular subgroups were identified, each of them characterized by a NOTCH pathway mutated gene (Fig. 1A). The first cluster was defined by NOTCH2 and/or KLF2 mutations and was enriched in TNFAIP3 mutations and IGHV1-2*04 gene usage (Fig. 1A). The second cluster was defined by SPEN mutations, and was enriched in KMT2D and other epigenetic gene mutations (Fig. 1A). The third cluster was enriched in NOTCH1 mutations (Fig. 1A). By relative survival analysis, the NOTCH2/KLF2 cluster showed a lower survival compared to the matched general population, indicating a significant impact of the disease on patients' expected survival (Fig. 1B). Conclusions. The large sample size and inclusion of SMZL confirmed by spleen histopathology review allowed for precise estimation of the prevalence of KLF2 and NOTCH2 mutations in this lymphoma. Three molecular clusters were identified in SMZL, each of them containing a NOTCH pathway gene, supporting the relevance of NOTCH signaling in the pathogenesis of SMZL. Patients belonging to the NOTCH2/KLF2 cluster had a lower relative survival compared to the matched general population. Disclosures Traverse-Glehen: Astra Zeneca: Other: Travel; Takeda: Research Funding. Gomes da Silva:Roche: Other: Institution's payment for consultancy, Travelling support; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Celgene: Other: Travelling support; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Institution's payment for consultancy, Travelling support; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Research Funding. Ladetto:Celgene: Honoraria; Jannsen: Honoraria; Acerta: Honoraria; Abbvie: Honoraria; Sandoz: Honoraria; Roche: Honoraria. Rambaldi:Pfizer: Consultancy; Novartis: Consultancy; Omeros: Consultancy; Italfarmaco: Consultancy; Amgen Inc.: Consultancy; Roche: Consultancy; Celgene: Consultancy. Vitolo:Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Zinzani:MSD: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Speakers Bureau. Gaidano:Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Salles:Servier: Honoraria; Abbvie: Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Amgen: Honoraria; BMS: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board, Research Funding; Acerta: Honoraria; Janssen: Honoraria, Other: Advisory Board; Merck: Honoraria; Pfizer: Honoraria; Morphosys: Honoraria; Gilead: Honoraria, Other: Advisory Board; Epizyme: Honoraria; Servier: Honoraria, Other: Advisory Board; Takeda: Honoraria. Zucca:Celltrion: Consultancy; AstraZeneca: Consultancy.

Description: Key Points Development of treatment resistance through further somatic mutations may occur in Erdheim-Chester disease during BRAF inhibition. Combinatorial BRAF/MEK inhibition may be beneficial in treatment-resistant ECD harboring a BRAFV600E and further MAPK-activating mutations.

Description: Introduction: RNA expression profiling using Next Generation Sequencing (NGS) provides important and reproducible information on expression levels of various genes. We studied the expression levels of MYC and BCL2 in patients with diffuse large B-Cell lymphoma (DLBCL) using NGS targeted RNA sequencing. We correlated levels of expression of these genes with outcome. Methods: RNA extracted from 441 FFPE samples with DLBC lymphoma and sequenced targeting 1408 genes. The RNA sequencing is based on hybrid capture and the number of reads ranged from 5 to 10 million. RNA quantification was performed using Cufflinks. The RNA levels were normalized to PAX5 mRNA levels. Of these cases, 380 were subclassified as ABC or GCB using expression profiling. The rest were classified as "undetermined", therefore were not included in further analysis. All patients were treated with R-CHOP. Results: The expression of MYC and BCL2 mRNA was slightly higher in ABC as compared with GCB (P=0.01 and P=0.02, respectively). However, in the GCB subtype, patients with MYC expression above the median showed significantly shorter survival as compared with those below the median (P=0.0007, Log-rank test). In contrast, there was no significant difference in survival using median of MYC expression as cutoff in patients classified as ABC subtype (P=0.38). Using upper 15% cut-off point for BCL2 mRNA expression, GCB patients with high BCL2 expression had significantly shorter survival (P=0.005). In contrast, there was no significant difference in survival between high and low BCL2 expression groups in the ABC subtype (P=0.1). When both MYC and BCL2 are considered, patients with high expression of both BCL2 and MYC (double-RNA expression) had significantly shorter survival as compared with patients with low expression of both MYC and BCL2 (P=0.0009) when both GCB and ABC groups are considered. Patients with high BCL2 expression also had poor survival similar to those double-RNA expression. Considering only patients with GCB, high expressor of both MYC and BCL2 had significantly worse outcome (P=0.0015) as compared with low expressors of both MYC and BCL2, but patients with high BCL2 also had significantly poor outcome as compared to low expressor of both MYC and BCL2 (P=0.0005). In contrast, there was no difference in survival for high or low MYC and BCL2 expressor in the ABC group. Conclusion: The data support the concept that in DLBCL, MYC and BCL2 mRNA expression levels are clinically relevant in GCB, but not ABC subtype. Furthermore, targeted RNA sequencing might provide a reliable and practical objective approach for the subclassification of DLBCL and determining double-RNA expression lymphoma. Figure Disclosures Albitar: Genomic Testing Cooperative: Employment, Equity Ownership. Tam:Takeda: Consultancy; Paragon Genomics: Consultancy. Hsi:Abbvie: Research Funding; Eli Lilly: Research Funding; Cleveland Clinic&Abbvie Biotherapeutics Inc: Patents & Royalties: US8,603,477 B2; Jazz: Consultancy. Piris:Calgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees, Research Funding. Kantarjian:Immunogen: Research Funding; BMS: Research Funding; Cyclacel: Research Funding; Pfizer: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Ariad: Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria; AbbVie: Honoraria, Research Funding; Astex: Research Funding; Jazz Pharma: Research Funding; Novartis: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Research Funding.

Description: Key Points The sphingosine-1-phosphate receptor 2 is a bona fide tumor suppressor and transcriptionally regulated by the TGF-β/TGF-βR2/SMAD1 axis. The aberrant loss of SMAD1 expression is very common in DLBCL and provides a proliferative advantage to B cells in vitro and in vivo.

Description: Metabolic reprogramming is a hallmark of cancer. MYC oncoproteins control many aspects of this response, by inducing the expression of genes involved in mitochondrial biogenesis, glycolysis, glutaminolysis and amino acid transport. This coordinated response allows cancer cells to meet the demands for macromolecules and energy necessary to sustain the anabolic state. Normal cells adapt to nutrient-limiting conditions, such as amino acid (AA) starvation, by activating the autophagy-lysosomal pathway that is necessary for the maintenance of amino acid pools and for providing other building blocks (e.g., ATP) that are needed for cell survival. Surprisingly, our ex vivo and in vivo studies of premalignant and neoplastic MYC-expressing B cells of Eμ-Myc transgenic mice, and of human MYC-driven B cell lymphoma (e.g., Burkitt lymphoma), revealed that MYC suppresses the catabolic autophagy-lysosomal pathway, and that, accordingly, Myc-expressing premalignant and neoplastic B cells are exquisitely sensitive to AA starvation. For example, analyses of the effects of low (6%) versus high (20%) protein diets revealed that limiting AA pools in vivo selectively reduces the numbers of circulating premalignant Eμ-Myc B220+ B cells without affecting B cell numbers in wild type littermate mice. Thus, MYC-driven tumor cells are unable to sufficiently adapt to a state of nutrient deprivation (Figure 1). Expression analyses revealed that this MYC suppresses the autophagy-lysosomal system by transcriptionally repressing genes that encode regulators and components of this pathway, and that this response is a hallmark of human malignancies with MYC involvement. Further, suppressing these genes has functional consequences, where MYC provokes marked reductions in autophagic flux that lead to marked increases in the levels of cargo such as p62/Sequestrin that are normally degraded by this pathway. A master regulator of autophagy and lysosomal biogenesis is TFEB that, like MYC, functions as a basic helix-loop-helix leucine zipper transcription factor and shares a similar DNA recognition sequence. Our studies suggest that MYC blocks TFEB function at three levels. First, MYC can directly repress TFEB transcription. Second, MYC can directly repress TFEB transcription targets by competing with TFEB for binding to the promoter-regulatory regions of autophagy-lysosome gene targets. Third, MYC-expressing B cells have activated mTORC1, which phosphorylates TFEB and blocks its nuclear localization. Notably, forced reactivation of the autophagy-lysosomal pathway via inducible expression of a of a constitutively active (mTORC1-resistant and nuclear) form of TFEB (TFEBS211A) disables the malignant state, where TFEBS211A triggers cell cycle arrest and senescence of both mouse and human MYC-driven lymphomas ex vivo, and compromises tumorigenic potential in vivo. Thus, TFEB acts as tumor suppressor for MYC-driven malignancies. We hypothesized that MYC-driven tumor cells compensate for the reductions in the autophagy pathway and maintain AA homeostasis by activating compensatory mechanisms, including AA transport and the proteasome. In support of this notion, the expression of AA transporters and components of the proteasome, and AA transport and proteasome activity, are markedly augmented in premalignant and neoplastic MYC-expressing B cells. Accordingly, MYC-expressing B cells are exquisitely sensitive to treatment with proteasome inhibitors. Collectively, these findings suggest that MYC drives the anabolic state by suppressing the catabolic autophagy-lysosomal pathway, and that to maintain AA pools MYC-driven cancer cells up-regulate AA transport and the proteasome. This scenario provides attractive opportunities for combination therapies that should disable MYC-driven malignancies, including protein-restricted diets and proteasome and TORC1. Figure 1. Premalignant Eμ-Myc and wild type (WT) littermates were treated for one week with low (6%) and high (20%) protein diets and B220+ cell numbers in peripheral blood were assessed. Figure 1. Premalignant Eμ-Myc and wild type (WT) littermates were treated for one week with low (6%) and high (20%) protein diets and B220+ cell numbers in peripheral blood were assessed. Disclosures No relevant conflicts of interest to declare.

Description: Key Points BH3-only proteins Bim and Bmf jointly coregulate developmental cell death. Bim and Bmf act as a barrier against autoimmunity and malignant disease.